This work was supported by the National Natural Science Foundation of China (32070502, 31601697, 32072419 and the China Postdoctoral Science Foundation (2020M672205).

1. Target site selection and sgRNA design
<ol>
	<li>Collect as much sequence information as possible for the gene of interest through literature research and/or searching for the mRNA or coding DNA sequence (CDS) of the gene at NCBI and locust database24.</li>
	<li>Compare the sequence of the interested gene to its genomic DNA sequence to distinguish the exon and intron regions.</li>
	<li>Select a candidate region for target site design based on the research purpose. Design primer pairs to amplify the candidate region fragment and verify its wild-type sequence by sequencing analysis of the PCR product. 
	NOTE: There are different strategies for the selection of the candidate target region. For example, in most gene/protein function research, use the exon fragment close to its start codon as the candidate region to ensure that the whole protein/RNA function is lost after the genome editing (i.e., gene knock-out). While for the function analysis of a specific domain, select the candidate region at the beginning (or both ends) of the domain.</li>
	<li>Use online resources such as the E-CRISP Design25 to search for potential target sites in the candidate target region.
	<ol>
		<li>Select &#34;Drosophila melanogaster BDGP6&#34; (or any other insect) in the drop-down list of reference genomes and choose &#34;Input is FASTA sequence&#34; followed by pasting the sequence of target region into the textbox (in FASTA format).</li>
		<li>Start the application by pressing &#34;Start sgRNA search&#34; with &#34;medium&#34; and &#34;single design&#34; to get the possible target sites. Select 1-3 potential targets from the results based on their predicted scores and design the corresponding sgRNAs accordingly. 
		NOTE: There are many more online platforms for CRISPR design, such as CRISPOR, CHOPCHOP, CRISPRdirect, ZiFiT, and so on (see Table of Materials). Some of them have the function of specificity check for the species whose genomic sequence data is in their databases. However, the genomic sequence of locusts has not been found on any online website. It is better to use more than one online tool for the CRISPR design in locusts. Comprehensively, consider the results from different websites and select the sgRNA from the results on the principle of highest specificity, highest efficiency, and least mismatch rate (Figure 1A,B).</li>
	</ol>
	</li>
</ol>
2. Synthesis and verification of the sgRNA&#160;&#160;in vitro
<ol>
	<li>Synthesize the sgRNA using sgRNA synthesis kits according to the manufacturer&#180;s manual (Table of Materials). This procedure usually includes three steps: DNA template amplification, in vitro transcription, and sgRNA purification21. Dilute the synthesized sgRNA with nuclease-free water to a storage concentration of 300 ng/&#181;L for further use.</li>
	<li>Amplify and purify the target gene fragment to serve as the substrate of the in vitro cleavage assay of the CRISPR/Cas9 system. Perform these steps following the manufacturer&#39;s instructions.</li>
	<li>Dilute the purchased Cas9 protein to 300 ng/&#181;L with nuclease-free water. Incubate 1 &#181;L of sgRNA (300 ng/&#181;L) and 1 &#181;L of Cas9 protein (300 ng/&#181;L) with 200 ng of purified target fragment in the cleavage buffer at 37 &#176;C for 1 h (10 &#181;L of total volume; see Table 1). Estimate the efficiency of sgRNA induced Cas9 cleavage by agarose gel electrophoresis (Figure 1C). Select the sgRNAs with high activity for the following microinjection. 
	NOTE: The results of agarose gel electrophoresis can be converted to grayscale images with image processing software and the cleavage efficiency is estimated according to the grayscale of the bands of speculated sizes.</li>
</ol>
3. Microinjection and culture of the eggs
<ol>
	<li>Prepare the injection needles by pulling glass capillaries with a micropipette puller (Table of Materials). Set the parameters as follows: Heat to 588, Pull to 90, Velocity to 60, and Time to 40. Grind the tip of the injection needle with a micro grinder (Table of Materials). 
	NOTE: Ideal needle tips are open and sharp-edged (Figure 2A). It is highly recommended to prepare additional needles for the experiments because needles are sometimes blocked or accidentally broken during injections.</li>
	<li>Mix 1 &#181;L of Cas9 protein (300 ng/&#181;L) with 1 &#181;L of the verified sgRNA (300 ng/&#181;L) together to obtain the RNPs for injection. Add 8 &#181;L of RNase-free sterile water to make the final volume of the mixture to 10 &#181;L. Mix the solution thoroughly and keep it on ice. 
	NOTE: The final concentrations of the Cas9 protein and sgRNAs can be optimized according to the editing results. Avoid repetitive freezing and thawing of the prepared RNP solution and immediate use is recommended.</li>
	<li>Rear the male and female adult locusts together at 30 &#176;C with a 16 (light):8 (dark) photoperiod and supply them sufficient fresh wheat seedlings as food. Observe these locusts daily and put an oviposition pot (a plastic flowerpot or cup filled with wet sterile sand) into the rearing cage for oviposition once the locusts mated. 
	NOTE: Usually, 100 pairs of adult locusts reared in a rearing cage (40 cm x 40 cm x 40 cm) are enough to generate eggs for knocking out a single gene. Culture additional locust pairs if more eggs are needed.</li>
	<li>Collect the freshly-laid egg pods from the oviposition pot and wait for about 30 min for egg tanning. Gently isolate the eggs from the egg pods in water using a fine brush and wash them with sterile water three times. Keep the eggs in a Petri dish and add sterile water to keep them moist. 
	NOTE: Tanning of the newly-laid eggs can significantly improve the mutant efficiency21.</li>
	<li>Fill an injection needle with the prepared RNP solution and load it to the micromanipulator (Table of Materials).
	<ol>
		<li>Set the microinjection parameters as follows: 300 hPa of the injection pressure (pi), 0.5 s of the injection time (ti), and 25 hPa of the compensation pressure (pc).</li>
		<li>Press the pedal to evaluate the volume of the injected solution. Adjust the injection pressure and injection time to ensure that the injection volume is about 50-100 nL. 
		NOTE: Exhaust the residual air in the needle to make the injection solution continuous and controllable as much as possible. The connection of the needle and the micromanipulator need to be tight.</li>
	</ol>
	</li>
	<li>Arrange the sterile eggs regularly on the injection pad (Figure 2B,C) and place the pad on the object table of the microscope (Figure 3A). Adjust the magnification of the microscope until the eggs are observed. Adjust the microinjection needle under the microscope until the injection tip can be seen and position it near the egg to be injected.</li>
	<li>Start the injection at a suitable height and an angle of 30-45 degrees. Insert the tip into the egg carefully near the micropyles of the egg (Figure 3B) and press the pedal to accomplish the injection. Retract the needle quickly and move the injection pad for injection of the next egg. 
	NOTE: A slight expansion of the egg during the microinjection should be observed. A small amount of cytoplasmic leakage at the pinhole is acceptable. Replace the injection needle with a new one or adjust the injection angle if the outflow of fluid is too much.</li>
	<li>Transfer the injected eggs to a culture dish (a Petri dish with a piece of moist filter paper), and place them in an incubator at 30 &#176;C. 
	NOTE: It will take about 13-14 days till nymphs hatch from these injected eggs (on condition that mutation of the target gene does not affect the development of the embryos) (Figure 3C). Inject at least 100 eggs to ensure a sufficient hatching and eclosion amount for knocking out a specific gene.</li>
</ol>
4. Mutation rate estimation and the screening of mutants
<ol>
	<li>Check the development of the injected eggs every day after the injection. Transfer injected eggs to a new culture dish every 24 h in the first 5 days.</li>
	<li>On the 6th day (or later) after injection, randomly pick and transfer 10 eggs into a tube and adequately grind the eggs with two steel balls at 60 Hz for 6 min using a grinder (see Table of Materials). Resuspend the debris with 1 mL of PBS. Transfer 5 &#181;L of the mixture into 45 &#181;L of 50 mM NaOH and lyse at 95 &#176;C for 5 min. Add 5 &#181;L of 1 M Tris-HCl (pH=8.0) into the lysis system to terminate the alkaline lysis reaction. 
	NOTE: Eggs can be picked out for alkaline lysis on any day after the last transfer and multiple eggs can be used as one sample depending on their development situation (e.g., five 6-day-old embryos can be used as one sample, and two 10-day-old embryos can be used as one sample). Sometimes, it is not feasible to get the genomic DNA of eggs using the alkaline lysis method. Commercial genomic DNA extraction kits can be used as an alternative method for isolating the genomic DNA from the eggs.</li>
	<li>Take 1 &#181;L of the lysis product as the PCR template to amplify the target gene fragment (Table 2 and Table 3) and send the PCR products for sequencing. Compare the sequencing results with the wild-type sequence to preliminarily evaluate whether the CRISPR/Cas9 system cleaved the target gene in vivo (Figure 4A). Allow the rest of the eggs for subsequent development on the condition that indels are detected at the embryonic stage. 
	NOTE: In this way, the mutation rate can be preliminarily estimated at the embryo stage. If no indels are found at this step, prepare some new sgRNAs for the target gene and repeat the knock-out protocol from step 2.1.</li>
	<li>Transfer the hatched nymphs to a rearing cage and culture them as described in step 3.3. When the nymphs developed to the fifth instar stage, separate them with plastic culture cups (1 nymph/cup). 
	NOTE: It usually takes about 25-35 days for the nymphs to develop to their fifth instar and the most obvious phenotype is that the wing buds extend to the fourth or fifth abdominal segments. Moreover, it is recommended to record the phenotypes of dead nymphs to predict the relationship between the target gene and phenotypes in further research.</li>
	<li>Cut off about 2 mm length of the antennae with dissecting scissors and lyse it using the alkaline lysis method (step 4.2). Analyze the target gene fragment sequence of these nymphs as described above (step 4.3) to identify the G0 mutants (Figure 4B). 
	&#8203;NOTE: The antennae cut for lysis can be a little longer and grinding or mincing the antennae is helpful for lysis although the antennae can be directly digested. Individuals with multiple peaks near the target site in the sequencing result are identified as positive mutants and allowed for subsequent development and mating.</li>
</ol>
5. Establishment and passaging of mutant lines
<ol>
	<li>Perform cross breeding using the G0 mutants and wild-type locusts (Figure 4B). Collect the oocysts and incubate these oocysts separately at 30 &#176;C. Use 3-6 developed eggs in each oocyst to detect mutations as described in steps 4.2 and 4.3. Keep mutation-positive oocysts for the subsequent development and abandon the mutation-negative oocysts. 
	NOTE: Mutation rate estimation at the embryonic stage can greatly accelerate the screening of G1 mutants. Usually, 3-6 developed eggs can be mixed as one sample for PCR-mediated genotyping as described above.</li>
	<li>Rear the G1 nymphs as described in step 4.4. Cut off about 2 mm length of the antennae to perform PCR-based genotyping as described in step 4.5 for detecting G1 heterozygotes. Perform TA-cloning according to the manufacturer&#39;s instructions (see Table of Materials) to identify their mutations. Perform in-cross using G1 heterozygotes with the same mutations to obtain G2 nymphs. 
	NOTE: Sanger sequencing of the PCR products can only provide information for finding out the heterozygotes, but not enough information for identifying the exact mutations. Thus, TA cloning using the PCR products is required to clearly identify the mutations and can promote the establishment of stable mutant lines.</li>
	<li>Rear the G2 nymphs till their fifth instar. Use the PCR-based genotyping described in step 5.2 to identify the homozygotes and/or heterozygotes that are suitable for further research and stable passaging (Figure 4B). 
	&#8203;NOTE: Pay attention to avoid mixing of homozygotes and heterozygotes. It is recommended to perform PCR-based genotyping in every generation to confirm the homozygosity and/or heterozygosity of each population. The number of locusts used for this check depends on the population size. Moreover, the population of locusts can be expanded by the in-cross strategy.</li>
</ol>
6. Egg cryopreservation and resuscitation
<ol>
	<li>Wash the eggs to be cryopreserved with sterile water and incubate them in a culture dish as described above (step 3.8) for 5-6 days. Gather these eggs together in the culture dish and cover them with filter paper fragments. Wrap the entire culture dish with a paraffin film.</li>
	<li>Keep the dish at 25 &#176;C for 2 days followed by another 2 days at a relatively lower temperature (13-16 &#176;C). Then, transfer the dish to a 6 &#176;C refrigerator. Add water into it every 2 weeks to provide a moist environment for the eggs (Figure 5A). 
	NOTE: The embryos are speculated to be at the katatrepsis stage after being developed for 5-6 days at 30 &#176;C26. Gradient cooling is helpful for cryopreservation of eggs (Table 5).</li>
	<li>For resuscitating the cryopreserved eggs, take the Petri dish out from the refrigerator and keep it at 25 &#176;C for 2 days. Place these eggs in a 30 &#176;C incubator until the nymphs hatch (Figure 5B). 
	NOTE: It is necessary to put the cryopreserved eggs at 25 &#176;C before transferring them to a 30 &#176;C incubator. The embryos can be cryopreserved for at least five months although the hatching rate and eclosion rate can decrease because of the cryopreservation (Table 5).</li>
</ol>

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The authors declare that they have no conflicts of interest.

Locusts have been among the most devastating pests to agriculture since the civilization of human beings23. CRISPR/Cas9-based genome editing technology is a powerful tool for providing better knowledge of the biological mechanisms in locusts as well as a promising pest control strategy. Thus, it is of great benefit to develop an efficient and easy-to-use method of CRISPR/Cas9-mediated construction of homozygous locust mutants. Although some great works have been reported and provided some basic wo...

Gene editing technologies could be used to introduce insertions or deletions into a specific genome locus to artificially modify the target gene on purpose1. In the past years, CRISPR/Cas9 technology has developed rapidly and has a growing scope of applications in various fields of life sciences2,3,4,5,6. The CRISPR/Cas9 system was discovered back in 19877, and widely found in bacteria and archaea. Further research indicated that it was a prokaryotic adaptive immune system that depends on the RNA-guided nuclease Cas9 to fight against phages8. The artificially modified CRISPR/Cas9 system mainly consists of two components, a single guide RNA (sgRNA) and the Cas9 protein. The sgRNA is made up of a CRISPR RNA (crRNA) complementary to the target sequence and an auxiliary trans-activating crRNA (tracrRNA), which is relatively conserved. When the CRISPR/Cas9 system is activated, the sgRNA forms a ribonucleoprotein (RNP) with the Cas9 protein and guides Cas9 to its target site via the base pairing of RNA-DNA interactions. Then, the double-strand DNA can be cleaved by the Cas9 protein and as a result, the double-strand break (DSB) emerges near the protospacer adjacent motif (PAM) of the target site9,10,11,12. To mitigate the damage caused by the DSBs, cells would activate comprehensive DNA damage responses to efficiently detect the genomic damages and initiate the repair procedure. There are two distinct repair mechanisms in the cell: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is the most common repair pathway that can repair DNA double-strand breaks quickly and prevents cell apoptosis. However, it is error-prone because of leaving small fragments of insertions and deletions (indels) near the DSBs, which usually results in an open reading frame shift and thus can lead to gene knock-out. In contrast, homologous repairment is quite a rare event. On the condition that there is a repair template with sequences homologous to the context of the DSB, cells would occasionally repair the genomic break according to the nearby template. The result of HDR is that the DSB is precisely repaired. Especially, if there is an additional sequence between the homologous sequences in the template, they would be integrated into the genome through HDR, and in this way, the specific gene insertions could be realized13.
With the optimization and development of the sgRNA structures and Cas9 protein variants, the CRISPR/Cas9-based genetic editing system has also been successfully applied in research of insects, including but not limited to Drosophila melanogaster, Aedes aegypti, Bombyx mori, Helicoverpa Armigera, Plutella xylostella, and Locusta migratoria14,15,16,17,18,19. To the best of the authors&#39; knowledge, although RNPs consisting of the Cas9 protein and in vitro transcribed sgRNA have been used for locust genome editing20,21,22, a systematic and detailed protocol for CRISPR/Cas9 ribonuclease mediated construction of homozygous mutants of the migratory locust is still lacking.
The migratory locust is an important agricultural pest that has a global distribution and poses substantial threats to food production, being especially harmful to gramineous plants, such as wheat, maize, rice, and millet23. Gene function analysis based on genome editing technologies can provide novel targets and new strategies for the control of migratory locusts. This study proposes a detailed method for knocking out migratory locust genes via the CRISPR/Cas9 system, including the selection of target sites and design of sgRNAs, in vitro synthesis and verification of the sgRNAs, microinjection and culture of eggs, estimation of mutation rate at the embryonic stage, detection of mutants as well as passage and preservation of the mutants. This protocol could be used as a basal reference for manipulation of the vast majority of locust genes and can provide valuable references for genome editing of other insects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10&#215;NEBuffer r3.1</td>
			<td>New England Biolabs</td>
			<td>B7030S</td>
			<td>&#160;The buffer of &#160;in vitro Cas9 cleavage assays</td>
		</tr>
		<tr>
			<td>2xEs Taq MasterMix (Dye)</td>
			<td>Cwbio</td>
			<td>CW0690</td>
			<td>For gene amplification</td>
		</tr>
		<tr>
			<td>2xPfu MasterMix (Dye)</td>
			<td>Cwbio</td>
			<td>CW0686</td>
			<td>For gene amplification</td>
		</tr>
		<tr>
			<td>CHOPCHOP</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://chopchop.cbu.uib.no/.</td>
		</tr>
		<tr>
			<td>CRISPOR</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://crispor.org.</td>
		</tr>
		<tr>
			<td>CRISPRdirect</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://crispr.dbcls.jp/.</td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>LIUYI BIOLOGY</td>
			<td>DYY-6D</td>
			<td>Separation of nucleic acid molecules of different sizes</td>
		</tr>
		<tr>
			<td>Eppendorf Tube</td>
			<td>Eppendorf</td>
			<td>30125177</td>
			<td>For sample collection, etc.</td>
		</tr>
		<tr>
			<td>Fine brushes</td>
			<td>Annigoni</td>
			<td>1235</td>
			<td>For cleaning and isolating eggs. Purchased online.</td>
		</tr>
		<tr>
			<td>Flaming/brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P97</td>
			<td>For making the microinjection needles</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Cwbio</td>
			<td>CW2302</td>
			<td>DNA recovery and purification</td>
		</tr>
		<tr>
			<td>Gel Imaging Analysis System</td>
			<td>OLYMPUS</td>
			<td>Gel Doc XR</td>
			<td>Observe the electrophoresis results</td>
		</tr>
		<tr>
			<td>GeneTouch Plus</td>
			<td>Bioer</td>
			<td>B-48DA</td>
			<td>For gene amplification</td>
		</tr>
		<tr>
			<td>Glass electrode capillary</td>
			<td>Gairdner</td>
			<td>GD-102</td>
			<td>For making&#160;injection needles with&#160;a&#160;micropipette Puller</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>MEMMERT</td>
			<td>INplus55</td>
			<td>For migratory locust embryo culture</td>
		</tr>
		<tr>
			<td>Metal bath</td>
			<td>TIANGEN</td>
			<td>AJ-800</td>
			<td>For heating the sample</td>
		</tr>
		<tr>
			<td>Micro autoinjector</td>
			<td>Eppendorf</td>
			<td>5253000068</td>
			<td>Microinjection of embryos early in development</td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>Allsheng</td>
			<td>MTV-1</td>
			<td>Used for mixing reagents</td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>NARISHIGE</td>
			<td>EG-401</td>
			<td>To ground the tip of injection needle</td>
		</tr>
		<tr>
			<td>Microloader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>For loading solutions into the injection needles.</td>
		</tr>
		<tr>
			<td>Micromanipulation system</td>
			<td>Eppendorf</td>
			<td>TransferMan 4r</td>
			<td>An altinative manipulation system for microinjection</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>cnoptec</td>
			<td>SZ780</td>
			<td>For microinjection</td>
		</tr>
		<tr>
			<td>Motor-drive Manipulator</td>
			<td>NARISHIGE</td>
			<td>MM-94</td>
			<td>For controling the position of the micropipette during the microinjection precedure</td>
		</tr>
		<tr>
			<td>Multi-Sample Tissue Grinder</td>
			<td>jingxin</td>
			<td>Tissuelyser-64</td>
			<td>Grind and homogenize the eggs</td>
		</tr>
		<tr>
			<td>ovipisition pot</td>
			<td>ChangShengYuanYi</td>
			<td>CS-11</td>
			<td>Filled with wet sterile sand for locust ovipositing in it. The oocysts are collected from this container. Purchased online.</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>ParafilmM</td>
			<td>PM996</td>
			<td>For wrapping the petri dishes.</td>
		</tr>
		<tr>
			<td>pEASY-T3 Cloning Kit</td>
			<td>TransGen Biotech</td>
			<td>CT301-01</td>
			<td>For TA cloning</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>NEST</td>
			<td>752001</td>
			<td>For culture and preservation of&#160; the eggs.</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf</td>
			<td>Research&#174;plus</td>
			<td>&#160;For sample loading</td>
		</tr>
		<tr>
			<td>plastic culture cup</td>
			<td></td>
			<td></td>
			<td>For rearing locusts seperately and any plastic cup big enough (not less than 1000 mL in volume) will do. Purchased online.</td>
		</tr>
		<tr>
			<td>Precision gRNA Synthesis Kit</td>
			<td>Thermo</td>
			<td>A29377</td>
			<td>For sgRNA synthesis</td>
		</tr>
		<tr>
			<td>Primer Premier</td>
			<td>PREMIER Biosoft</td>
			<td>Primer Premier 5.00</td>
			<td>For primer design</td>
		</tr>
		<tr>
			<td>SnapGene</td>
			<td>Insightful Science</td>
			<td>SnapGene&#174;4.2.4</td>
			<td>For analyzing&#160; sequences</td>
		</tr>
		<tr>
			<td>Steel balls</td>
			<td>HuaXinGangQiu</td>
			<td>HXGQ60</td>
			<td>For sample grinding.Purchased online.</td>
		</tr>
		<tr>
			<td>Tips</td>
			<td>bioleaf</td>
			<td>D781349</td>
			<td>&#160;For sample loading</td>
		</tr>
		<tr>
			<td>Trans DNA Marker II</td>
			<td>TransGen Biotech</td>
			<td>BM411-01</td>
			<td>Used to determine gene size</td>
		</tr>
		<tr>
			<td>TrueCut Cas9 Protein v2</td>
			<td>Thermo</td>
			<td>A36496</td>
			<td>Cas9 protein</td>
		</tr>
		<tr>
			<td>UniversalGen DNA Kit</td>
			<td>Cwbio</td>
			<td>CWY004</td>
			<td>For genomic DNA extraction</td>
		</tr>
		<tr>
			<td>VANNAS Scissors</td>
			<td>Electron Microscopy Sciences</td>
			<td>72932-01</td>
			<td>For cutting off the antennae</td>
		</tr>
		<tr>
			<td>Wheat</td>
			<td></td>
			<td></td>
			<td>To generate wheat seedlings as the food for locusts. Bought from local farmers.</td>
		</tr>
		<tr>
			<td>ZiFiT</td>
			<td></td>
			<td></td>
			<td>Online website for designing sgRNAs, http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx.</td>
		</tr>
	</tbody>
</table>

construction of homozygous mutants of migratory locust using crispr/cas9 technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This protocol contains the detailed steps for generating homozygous mutants of the migratory locusts with the RNP consisting of Cas9 protein and in vitro synthesized sgRNA. The following are some representative results of CRISPR/Cas9-mediated target gene knockout in locusts, including target selection, sgRNA&#160;synthesis and verification (Figure 1A), egg collection and injection, mutant screening and passaging, cryopreservation, and resuscitation of the homozygous eggs.
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Watch this Scientific Journal Video about Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology at JoVE.com

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

construction-homozygous-mutants-migratory-locust-using-crisprcas9

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JoVE Journal

Biology

1.9K Views.  Henan University. This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

Video: Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops worldwide. Since adult fruit flies have great flight capacity and females lay their eggs under the skins of fruit, insecticides requiring direct contact with the pest usually perform poorly in the field. With the development of molecular biological tools and high-throughput sequencing technology, many scientists are attempting to develop environmentally friendly pest management strategies. These include RNAi or gene editing-based pesticides that downregulate or silence genes (molecular targets), such as olfactory genes involved in searching behavior, in various insect pests. To adapt these strategies for Oriental fruit fly control, effective methods for functional gene research are needed. Genes with critical functions in the survival and reproduction of B. dorsalis serve as good molecular targets for gene knockdown and/or silencing. The CRISPR/Cas9 system is a reliable technique used for gene editing, especially in insects. This paper presents a systematic method for CRISPR/Cas9 mutagenesis of B. dorsalis, including the design and synthesis of guide RNAs, collecting embryos, embryo injection, insect rearing, and mutant screening. These protocols will serve as a useful guide for generating mutant flies for researchers interested in functional gene studies in B. dorsalis.

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.

In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive ...

Centromere-Associated Protein-E Gene Editing Using the CRISPR/Cas9 System

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Author Spotlight: Establishing CENP-E Knockout HeLa Cells &#8211; A Novel Approach to Study Kinesin-7 CENP-E Biology and its Inhibitors

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...